Dabrafenib is a multi-kinase inhibitor

Dabrafenib (Tafinlar®, GSK-2118436) is an approved inhibitor for the treatment of malignant melanoma in patients with a BRAF(V600E) mutation. Despite the general notion as selective BRAF(V600E) inhibitor²⁵⁰, Kinobeads profiling in cell mix not only revealed its known target BRAF but also showed binding to 47 other targets. Thereof, 30 proteins are kinases and show binding affinities below 1 µM. Figure 29a depicts the distribution of kinases across the kinome, the size of the dots is proportional to the potency of the inhibition. Kinase targets of Dabrafenib cannot only be found in the tyrosine kinase like branch where BRAF is located but are also distributed across the tyrosine kinase family, the CAMK and the CMGC group. Studies on Dabrafenib activity only report on selective BRAF inhibition with a few related kinases inhibited (Figure 29b)^{250, 251}. KinomeScan data published on the LINCS database (Dataset 20131, LINCS²¹¹) obtains similar binding results to the Kinobeads data. Here, off-targets were also identified in the TK and CMGC branch of the kinome (Figure 29c). This confirmed the obtained binding data as well as the suitability of Kinobeads for selectivity profiling.

As the discrepancy between binding data and known activity inhibition for Dabrafenib was so large, the additional targets were subjected to recombinant activity assays. These were performed at Reaction Biology Corporation, monitoring phosphorylation activity of recombinant kinases and radioisotope labeled ATP. In general, most of the identified kinases that bound to Dabrafenib also Figure 29: Dabrafenib target space. Kinome tree representation of Dabrafenib targets (courtesy of Cell Signaling Technologies). a) Targets identified in the Kinobeads screen. Size of the dots is proportional to affinity in nM. b) Reported activity [nM] for Dabrafenib and selected kinases in literature.c) Reported KinomeScan data for Dabrafenib (Dataset 20131, LINCS). Size of dots is proportional to % control after 10 µM compound treatment.

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showed reduction in activity of the respective kinase (Figure 30a, b). Besides binding to ARAF and BRAF in the proteomics assay, Dabrafenib could inhibit the activity of the whole RAF-family with IC50 values of 0.14 nM for ARAF, 0.63 nM for BRAF, 0.51 nM for RAF1 and 4.05 nM for the mutant version BRAF(V600E) in the recombinant panel. At these low doses, the effect on selective BRAF(V600E) inhibition in the clinic is supposedly due to the inhibition of the cancer driving mutation, whereas wildtype BRAF melanomas are not dependent on BRAF. Reduced activity could also be confirmed for the actin filament regulatory kinases LIMK1 and LIMK2, ULK1 involved in autophagy, as well as for RIPK2, RIPK3, SIK2, SIK3, ULK1 and ZAK, which take part in immune regulatory pathways. Binding inhibition of some members of the SRC-family (LCK, LYN, FYN, FGR, FRK) as well as other tyrosine kinases also led to loss of kinase activity. ABL1, IRAK1 and TGFBR1 showed the weakest EC50s and, thus, might only be fully inhibited upon higher dosage (Figure 30, Table 2).

Notably, the binding profile revealed several interactions with CDK-family members. Besides potent CDK16 and CDK17 binding, Dabrafenib could bind to all cyclin-dependent kinases directly involved in cell cycle progression (CDK1, CDK2, CDK4, CDK5 and CDK6, Figure 31b). For further investigation, Dabrafenib was also incubated with MALME 3M lysate, a melanoma cancer cell line expressing the BRAF(V600E) mutation. Subsequent Kinobeads pulldowns confirmed binding to CDK2, CDK4, CDK5 and CDK6 but not to CDK1 (Figure 31b).

In activity assays, CDK-activity is assessed with the help of a cyclin. Various CDK-cyclin combinations are available for testing representing known CDK-cyclin interactions in the cell. The potent Figure 30: Confirmation of Dabrafenib targets. a) Dose response curves of the Kinobeads competition experiment with Dabrafenib for several kinases. b) Recombinant kinase activity assays confirm inhibition of novel Dabrafenib targets. Besides ARAF, BRAF and RAF1, also several kinases involved in immune regulatory pathways (e.g. RIPK2, SIK2) and members of the SRC-family could be confirmed as targets.

57 inhibition of Dabrafenib on CDK16, a kinase involved in vesicle mediated transport and exocytosis, was confirmed in combination with cyclin Y. CDK4 in combination with CCND1 and D3, as well as CDK6 with CCND3 could also be reduced in their activity, even though with slightly less potent EC50s than anticipated from the Kinobeads pulldown. Interestingly, CDK6 together with CCND1 showed no inhibition (Figure 31c). The binding of Dabrafenib to CDK5 at 1 µM was also reflected in 3-5 µM EC50s in the activity assay for the respective CDK5-p25/p35 combinations. CDK1 inhibition could be partially confirmed by inhibition of CDK1 together with CCNE or CCNB1 but not with CCNA2.

Surprisingly, no CDK2-cyclin combination tested (CCNE, CCNO, CCN2) had reduced activity upon Dabrafenib treatment, but CDK2 alone was detected as target in both the Kinobeads and KinomeScan data.

As kinobeads pull down protein-protein complexes, also potential CDK-interacting cyclins were identified in the Dabrafenib screen. Contrary to other CDK inhibitors in the screen, only CCNB2 showed a dose response profile similar to their respective CDK interaction partner CDK1 in the cell mix and was also co-competed in the MALME-3M lysate, whereas CCNA2, CCNB1 and CCNE1/2 showed no effect. CCNA2 and CCNE1/2 are the known interaction partners of CDK2 in the cell cycle.

One can now speculate whether CDK2 interacts with CCNB2 (known to interact with CDK1), which could not be assessed in commercial activity assays, or whether it can bind to Dabrafenib alone or in combination with an unknown cyclin not detected in the screen and not available for testing in an activity assay. This prevented clear assessment of potential CDK inhibition by Dabrafenib.

Activity assays for Dabrafenib also revealed a general concordance between targets identified in a binding assay and affected activity (Table 2). Except for the CDK-targets, all targets identified in the binding assay also show impaired activity upon Dabrafenib treatment. Hence, Dabrafenib in fact needs to be considered as a multi-kinase inhibitor, with potential off-target toxicities because of multiple kinase inhibition.

Figure 31: Binding and activity assays for Dabrafenib and CDK. a) Kinobeads competition for several CDKs and their cyclins in cell mix. All CDKs show inhibition, whereas only CCNB1 shows a similar dose respons. b) Kinobeads competition of CDKs and cyclins in MALME-3M lysate. c) Recombinant kinase assays for CDK/cyclin pairs. The potent inhibition of CDK2 in the Kinobeads experiments could not be confirmed.

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Table 2: Binding and activity data for Dabrafenib. Kdapp and EC50 values for kinase targets of Dabrafenib in the Kinobeads screen (binding) and in recombinant kinase assays (activity, performed at Reaction Biology Corporation).